README - Chapter 3 (Results Chapter 1) Documentation 

Project Title 
Investigating the role of serotonergic receptor subtypes in the modulation of cell-specific immune function

Dataset DOI: https://doi.org/10.5258/SOTON/PG/D228

Author
Jamie Thomas

Institution 
University of Southampton 

Supervisor 
Professor Diego Gomez-Nicola
Dr Max Ahmed 
Dr Gary Gilmore

Date
April 2026

Overview
This folder contains the dataset(s), supporting materials, and analysis files used in Chapter 3 of the PhD thesis. The purpose of this README is to document the contents, structure, and usage of the data for transparency, reproducibility, and future reference.

Purpose
This data was used to investigate the expression of serotonergic receptor subtypes in immune cells. Both primary and secondary data were used to address this research question. 

Structure and Details

1. ImmGen Data
Data Files (3) - Excel 
Data obtained from the ImmGen Project Database
3 files represent 3 separate dates that the data was obtained, to ensure it was up to date 
Key variable = level of expression of the  serotonergic system across murine and human immune cells 
Normalised data extracted, refined for focus, and used to generate heatmaps 
Heatmaps used to visualise the data 
Note: not our data so not specific to our experimental aim, variable levels of biological replicates used, limited human data.

2. R Analysis 
Data Folders (3) - Data, Additional
'Data' contains figure extracts from R workflow for the dataset utilised in the R analysis (within Figure Examples), alongside the R history, saved Seurat objects and directory for the work (within Data). 'Additional' contains general notes and those related to the seurat pipeline. 
Key variable = level of expression of the  serotonergic system across murine immune cells
Data extracted, analysed in R by making seurat object and following seurat pipeline - figures extracted for visualisation. 
Feature plots used to visualise data
Note: threshold for serotonergic system detection was not met so expression could not be identified

3. PCR - Spleen 
Data Files (7) - Excel 
Data obtained from RT-PCR experiments 
7 Files represent the 5 genes of interest, the housekeeper gene, and a summary document
Key variable = level of serotonergic receptor expression in spleen tissue
CT values obtained from RT-PCR, delta-deltaCT used to analyse the level of expression 
Graphs used to visualise the relative expression of genes of interest
Note: signal in whole spleen samples appeared to be too weak for the expression of serotonergic receptors 

4. PCR - Spleen Cells
Data Files (2) - Excel/PRISM and Data Folders (4) PCR 0.35 Threshold, PCR 0.3 Threshold, Melt Curve, Additional Documents
'PCR-Genes' provides a summary of the results, with 'PCR-Graphs and Stats' being the associated analysis and graphs. Both 'PCR 0.35 Threshold' and 'PCR 0.3 Threshold' folders contain the raw data for the expression of genes of interest using the corresponding thresholds, with 0.35 being selected for the final data. 'Melt Curves' correspond to the primers used in the experiments. The 'Additional Documents' provide notes on the primers and how they were selected.
CT values obtained from RT-PCR, delta-deltaCT used to analyse the level of expression
Graphs used to visualise the relative expression of genes of interest


Contact
Name: Jamie Thomas
Email: jlt1n21@soton.ac.uk 